Real-time PCR

Real-time PCR analysis and calculation of target gene expression following the methods described previously (Wang et al., 2008; Kang et al., 2013) with modifications. Each PCR contained 8 μL cDNA (100 × dilution), 2 μL of either Tnfxyd8 or β-actin (internal control) primer mixture (100 nM), and 10 μL SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA), using the ABI PRISM 7300 Real-Time PCR System (Applied Biosystems). Primer sequences are shown in Table S1. Melting curve analysis and electrophoresis were performed to confirm the specificity of the amplification. For each unknown sample, relative Tnfxyd8 expression was obtained using the formula 2^∧ −[(Ct_Tnfxyd8,n−Ct_β−actin,n)−(Ct_Tnfxyd8,c−Ct_β−actin,c)], where Ct corresponded to the threshold cycle number.